Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 10 de 10
Filtrar
1.
J Am Chem Soc ; 141(20): 8064-8067, 2019 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-31034218

RESUMO

Transition state stabilization is essential for rate acceleration of enzymatic reactions. Despite extensive studies on various transition state structures of enzymes, an intriguing puzzle is whether an enzyme can accommodate multiple transition states (TSs) to catalyze a chemical reaction. It is experimentally challenging to study this proposition in terms of the choices of suitable enzymes and the feasibility to distinguish multiple TSs. As a paradigm with the protein lysine methyltransferase (PKMT) SET7/9 paired with its physiological substrates H3 and p53, their TSs were solved with experimental kinetic isotope effects as computational constraints. Remarkably, SET7/9 adopts two structurally distinct TSs, a nearly symmetric SN2 and an extremely early SN2, for H3K4 and p53K372 methylation, respectively. The two TSs are also different from those previously revealed for other PKMTs. The setting of multiple TSs is expected to be essential for SET7/9 and likely other PKMTs to act on broad substrates with high efficiency.


Assuntos
Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , S-Adenosilmetionina/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Catálise , Histona-Lisina N-Metiltransferase/química , Histonas/química , Humanos , Cinética , Lisina/química , Lisina/metabolismo , Metilação , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional , S-Adenosilmetionina/química , Proteína Supressora de Tumor p53/química
2.
Proc Natl Acad Sci U S A ; 113(52): E8369-E8378, 2016 12 27.
Artigo em Inglês | MEDLINE | ID: mdl-27940912

RESUMO

Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (SN2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MS-based method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-l-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13C KIE of 1.04, an inverse intrinsic α-secondary CD3 KIE of 0.90, and a small but statistically significant inverse CD3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical SN2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steady-state kinetics with the SAM analog Se-adenosyl-l-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.


Assuntos
Histona-Lisina N-Metiltransferase/química , Isótopos/química , Ligação Competitiva , Catálise , Ciclo Celular , Simulação por Computador , Histonas/química , Humanos , Cinética , Lisina/química , Metilação , Modelos Moleculares , Modelos Teóricos , Metástase Neoplásica , Peptídeos/química , Estrutura Secundária de Proteína , S-Adenosilmetionina/química , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato
3.
Bioorg Med Chem ; 24(1): 12-9, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26672511

RESUMO

Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphoproteins in normal and diseased states is critical to fully characterize cell signaling. Towards phosphoprotein analysis tools, our lab reported kinase-catalyzed labeling where γ-phosphate modified ATP analogs are utilized by kinases to label peptides or protein substrates with a functional tag. In particular, the ATP-biotin analog was developed for kinase-catalyzed biotinylation. However, kinase-catalyzed labeling has been tested rigorously with only a few kinases, preventing use of ATP-biotin as a general tool. Here, biotinylation experiments, gel or HPLC-based quantification, and kinetic measurements indicated that twenty-five kinases throughout the kinome tree accepted ATP-biotin as a cosubstrate. With this rigorous characterization of ATP-biotin compatibility, kinase-catalyzed labeling is now immediately useful for studying phosphoproteins and characterizing the role of phosphorylation in various biological events.


Assuntos
Biotina/análogos & derivados , Proteínas Quinases/química , Biotina/química , Biotinilação , Humanos , Cinética , Simulação de Acoplamento Molecular , Fosforilação
4.
Bioorg Med Chem ; 22(5): 1620-5, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24529309

RESUMO

Kinase-catalyzed protein phosphorylation is an important post-translational modification that regulates a variety of cellular functions. Identification of the many substrates of a specific kinase is critical to fully characterize cell biology. Unfortunately, kinase-substrate interactions are often transient, which makes their identification challenging. Here, the transient kinase-substrate complex was stabilized by covalent crosslinking using γ-phosphate modified ATP analogs. Building upon prior use of an ATP-aryl azide photocrosslinking analog, we report here the creation of an ATP-benzophenone photocrosslinking analog. ATP-benzophenone displayed a higher conversion percentage but more diffuse crosslinking compared to the ATP-aryl azide analog. A docking study was also performed to rationalize the conversion and crosslinking data. In total, the photocrosslinking ATP analogs produced stable kinase-substrate complexes that are suitable for future applications characterizing cell signaling pathways.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Fosforilação/fisiologia , Proteínas Quinases/metabolismo , Trifosfato de Adenosina/metabolismo , Humanos , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Especificidade por Substrato
5.
Chembiochem ; 14(3): 381-7, 2013 Feb 11.
Artigo em Inglês | MEDLINE | ID: mdl-23335220

RESUMO

Kinase-catalyzed protein phosphorylation is involved in a wide variety of cellular events. Development of methods to monitor phosphorylation is critical to understand cell biology. Our lab recently discovered kinase-catalyzed biotinylation, where ATP-biotin is utilized by kinases to label phosphopeptides or phosphoproteins with a biotin tag. To exploit kinase-catalyzed biotinylation for phosphoprotein purification and identification in a cellular context, the susceptibility of the biotin tag to phosphatases was characterized. We found that the phosphorylbiotin group on peptide and protein substrates was relatively insensitive to protein phosphatases. To understand how phosphatase stability would impact phosphoproteomics research applications, kinase-catalyzed biotinylation of cell lysates was performed in the presence of kinase or phosphatase inhibitors. We found that biotinylation with ATP-biotin was sensitive to inhibitors, although with variable effects compared to ATP phosphorylation. The results suggest that kinase-catalyzed biotinylation is well suited for phosphoproteomics studies, with particular utility towards monitoring low-abundance phosphoproteins or characterizing the influence of inhibitor drugs on protein phosphorylation.


Assuntos
Fosfoproteínas Fosfatases/metabolismo , Fosfoproteínas/metabolismo , Proteínas Quinases/metabolismo , Proteômica , Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Biocatálise , Biotina/química , Biotina/metabolismo , Biotinilação , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas/química , Fosforilação/efeitos dos fármacos , Proteínas Quinases/química , Especificidade por Substrato
6.
Bioconjug Chem ; 23(12): 2386-91, 2012 Dec 19.
Artigo em Inglês | MEDLINE | ID: mdl-23116557

RESUMO

Kinase-catalyzed protein phosphorylation is an important biochemical process involved in cellular functions. We recently discovered that kinases promiscuously accept γ-modified ATP analogues as cosubstrates and used several ATP analogues as tools for studying protein phosphorylation. Herein, we explore the structural requirements of γ-modified ATP analogues for kinase compatibility. To understand the influence of linker length and composition, a series of ATP analogues was synthesized, and the efficiency of kinase-catalyzed labeling was determined by quantitative mass spectrometry. This study on factors influencing kinase cosubstrate promiscuity will enable design of ATP analogues for a variety of kinase-catalyzed labeling reactions.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Aminas/química , Proteínas Quinases Dependentes de AMP Cíclico/química , Peptídeos/química , Coloração e Rotulagem/métodos , Sítios de Ligação , Catálise , Cinética , Espectrometria de Massas , Simulação de Acoplamento Molecular , Fosforilação , Relação Estrutura-Atividade , Especificidade por Substrato
7.
Nat Biotechnol ; 40(8): 1210-1219, 2022 08.
Artigo em Inglês | MEDLINE | ID: mdl-35288668

RESUMO

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.


Assuntos
Processamento Pós-Transcricional do RNA , Transcriptoma , Animais , Humanos , Mamíferos/genética , Metilação , RNA/genética , RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/genética , Transcriptoma/genética
8.
Cell Rep ; 36(4): 109421, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34320342

RESUMO

Mitogen-activated protein kinases (MAPKs) are inactivated by dual-specificity phosphatases (DUSPs), the activities of which are tightly regulated during cell differentiation. Using knockdown screening and single-cell transcriptional analysis, we demonstrate that DUSP4 is the phosphatase that specifically inactivates p38 kinase to promote megakaryocyte (Mk) differentiation. Mechanistically, PRMT1-mediated methylation of DUSP4 triggers its ubiquitinylation by an E3 ligase HUWE1. Interestingly, the mechanistic axis of the DUSP4 degradation and p38 activation is also associated with a transcriptional signature of immune activation in Mk cells. In the context of thrombocytopenia observed in myelodysplastic syndrome (MDS), we demonstrate that high levels of p38 MAPK and PRMT1 are associated with low platelet counts and adverse prognosis, while pharmacological inhibition of p38 MAPK or PRMT1 stimulates megakaryopoiesis. These findings provide mechanistic insights into the role of the PRMT1-DUSP4-p38 axis on Mk differentiation and present a strategy for treatment of thrombocytopenia associated with MDS.


Assuntos
Diferenciação Celular , Fosfatases de Especificidade Dupla , Megacariócitos , Fosfatases da Proteína Quinase Ativada por Mitógeno , Adulto , Animais , Criança , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Arginina/metabolismo , Linhagem Celular , Fosfatases de Especificidade Dupla/metabolismo , Estabilidade Enzimática , Células HEK293 , Sistema de Sinalização das MAP Quinases , Megacariócitos/citologia , Megacariócitos/enzimologia , Metilação , Camundongos Endogâmicos C57BL , Fosfatases da Proteína Quinase Ativada por Mitógeno/metabolismo , Síndromes Mielodisplásicas/enzimologia , Síndromes Mielodisplásicas/patologia , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Poliubiquitina/metabolismo , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/metabolismo , Proteólise , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/metabolismo , Ubiquitinação
10.
Curr Protoc Chem Biol ; 4(1): 83-100, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-25177527

RESUMO

Kinase-catalyzed protein phosphorylation plays an essential role in a variety of biological processes. Methods to detect phosphoproteins and phosphopeptides in cellular mixtures will aid in cell biological and signaling research. Our laboratory recently discovered the utility of γ-modified ATP analogues as tools for studying phosphorylation. Specifically, ATP-biotin can be used for labeling and visualizing phosphoproteins from cell lysates. Because the biotin tag is suitable for protein detection, the biotinylation reaction can be applied to multiple phosphoproteomics applications. Herein we report a general protocol for labeling phosphopeptides and phosphoproteins in biological samples using kinase-catalyzed biotinylation.

SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA